WO2015006657A2 - Méthode de diagnostic et de pronostic du cancer - Google Patents

Méthode de diagnostic et de pronostic du cancer Download PDF

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Publication number
WO2015006657A2
WO2015006657A2 PCT/US2014/046294 US2014046294W WO2015006657A2 WO 2015006657 A2 WO2015006657 A2 WO 2015006657A2 US 2014046294 W US2014046294 W US 2014046294W WO 2015006657 A2 WO2015006657 A2 WO 2015006657A2
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cancer
level
sample
biomarkers
individual
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PCT/US2014/046294
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English (en)
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WO2015006657A3 (fr
WO2015006657A8 (fr
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Majda HAZNADAR
Ewy MATHE
Curtis Craig Harris
Andrew D. PATTERSON
Soumen MANN
Frank Gonzalez
Kristopher KRAUSZ
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The Unitetd States Of America, As Represented By The Secretary, Department Of Health And Human Services
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Application filed by The Unitetd States Of America, As Represented By The Secretary, Department Of Health And Human Services filed Critical The Unitetd States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority to EP14745047.2A priority Critical patent/EP3019871B1/fr
Priority to US14/903,706 priority patent/US10393745B2/en
Publication of WO2015006657A2 publication Critical patent/WO2015006657A2/fr
Publication of WO2015006657A3 publication Critical patent/WO2015006657A3/fr
Publication of WO2015006657A8 publication Critical patent/WO2015006657A8/fr
Priority to US16/412,003 priority patent/US11555818B2/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/5743—Specifically defined cancers of skin, e.g. melanoma
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to the use of specific biomarkers in the detection of cancer and the prediction of the prognosis of cancer patients.
  • Methods of the present invention relate to the use of specific biomarkers to detect the presence of cancer in an individual.
  • the disclosed methods are also useful for determining the prognosis of an individual known to have cancer.
  • methods of the present invention may generally be accomplished by determining the levels of one or more specific biomarkers, disclosed herein, within an individual. Alterations in these levels relative to the levels of the same one or more biomarkers in individuals known to be free of cancer are indicative of the presence of cancer.
  • One embodiment of the present invention is a method for the detection of cancer, comprising determining the level of at least two compounds selected from the group consisting of creatine riboside, metabolite 561+, Cortisol sulfate and N-acetylneuraminic acid, in a sample obtained from an individual, wherein elevated levels of the at least two compounds indicates the presence of cancer.
  • the method comprises determining the level of at least three compounds selected from the group consisting of creatine riboside, metabolite 561+, Cortisol sulfate and N-acetylneuraminic acid in a sample obtained from an individual, wherein elevated levels of the at least three compounds indicates the presence of cancer.
  • the method comprises determining the level of creatine riboside, metabolite 561+, Cortisol sulfate and N-acetylneuraminic acid in a sample obtained from an individual, wherein elevated levels of all four compounds indicates the presence of cancer.
  • the cancer is lung cancer.
  • the cancer comprises adenocarcinoma.
  • the cancer comprises squamous cell carcinoma.
  • the sample is body tissue.
  • the sample is a body fluid.
  • the sample is urine.
  • the sample is selected from the group consisting of blood, serum and plasma.
  • One embodiment of the present invention is a method for the detection of cancer comprising determining the level of one or more compounds selected from the group consisting of creatine riboside and metabolite 561+, in a sample from an individual, wherein elevated levels of creatine riboside and/or metabolite 561+ indicate the presence of cancer.
  • the method comprises determining the levels of creatine riboside and metabolite 561+, in a sample, wherein elevated levels of creatine riboside and metabolite 561+ indicate the presence of cancer.
  • the cancer is lung cancer.
  • the cancer comprises adenocarcinoma.
  • the cancer comprises squamous cell carcinoma.
  • the sample is body tissue.
  • the sample is a body fluid.
  • the sample is urine.
  • the sample is selected from the group consisting of blood, serum and plasma.
  • One embodiment of the present invention is a method for the detection of lung cancer comprising determining the level of Cortisol sulfate in a sample obtained from an individual, wherein an elevated level of Cortisol sulfate indicates the presence of lung cancer.
  • the cancer comprises adenocarcinoma.
  • the cancer comprises squamous cell carcinoma.
  • the sample is body tissue.
  • the sample is a body fluid.
  • the sample is urine.
  • the sample is selected from the group consisting of blood, serum and plasma.
  • One embodiment of the present invention is a method for the detection of lung cancer, comprising determining the level of N-acetylneuraminic acid in urine from an individual, wherein an elevated level of urinary N-acetylneuraminic acid indicates the presence of lung cancer.
  • the N-acetylneuraminic acid is N-acetylneuraminic acid.
  • the cancer comprises adenocarcinoma. In one embodiment, the cancer comprises squamous cell carcinoma.
  • One embodiment of the present invention is a method for monitoring the efficacy of a cancer treatment, the method comprising: a) determining the level of one or more biomarkers selected from the group consisting of creatine riboside, metabolite 561+, Cortisol sulfate and N-acetylneuraminic acid in a sample patient having cancer to obtain a pre-treatment level of the one or more biomarkers;
  • the method comprises determining the level of two or more compounds selected from the group consisting of creatine riboside, metabolite 561+, Cortisol sulfate and N-acetylneuraminic acid in the sample. In one embodiment, the method comprises determining the level of three or more compounds selected from the group consisting of creatine riboside, metabolite 561+, Cortisol sulfate and N-acetylneuraminic acid in the sample.
  • the method comprises determining the level of creatine riboside, metabolite 561+, Cortisol sulfate and N-acetylneuraminic acid in the sample.
  • the cancer is lung cancer.
  • the cancer comprises adenocarcinoma.
  • the cancer comprises squamous cell carcinoma.
  • the sample is body tissue.
  • the sample is a body fluid.
  • the sample is urine.
  • the sample is selected from the group consisting of blood, serum and plasma.
  • One embodiment of the present invention is a method for predicting the prognosis of an individual having cancer, the method comprising determining the level of at least one biomarker selected from the group consisting of creatine riboside, Cortisol sulfate, metabolite 561+ and N-acetylneuraminic acid, in a sample from the individual, wherein an elevated level of the at least one biomarker is indicative of the prognosis of the individual.
  • the method comprises determining the level of two or more compounds selected from the group consisting of creatine riboside, metabolite 561+, Cortisol sulfate and N- acetylneuraminic acid in the sample.
  • the method comprises determining the level of three or more compounds selected from the group consisting of creatine riboside, metabolite 561+, Cortisol sulfate and N-acetylneuraminic acid in the sample. In one embodiment the method comprises determining the level of creatine riboside, metabolite 561+, Cortisol sulfate and N-acetylneuraminic acid in the sample.
  • the cancer is lung cancer. In one embodiment, the cancer comprises adenocarcinoma. In one embodiment, the cancer comprises squamous cell carcinoma. In one embodiment, the sample is body tissue. In one embodiment, the sample is a body fluid. In one embodiment, the sample is urine.
  • the sample is selected from the group consisting of blood, serum and plasma.
  • an elevated level of the at least one biomarker indicates a reduced survival time relative to a cancer patient in whom the level of the at least one biomarker is not elevated.
  • elevated levels of creatine riboside and metabolite 561+ indicate a reduced survival time relative to a cancer patient in whom the levels of creatine riboside and metabolite 561+ are not elevated.
  • elevated levels of creatine riboside, metabolite 561+, and N-acetylneuraminic acid indicate a reduced survival time relative to a cancer patient in whom the levels of creatine riboside, metabolite 561+ and N-acetylneuraminic acid are not elevated
  • NSCLC non-small cell lung cancer
  • the presence of an ALK or EGFR mutation indicates a responsive tumor to targeted therapies and longer survival (Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib.
  • Urine is gaining increasing interest as a biospecimen for detecting cancer biomarkers (Schmidt C. Urine biomarkers may someday detect even distant tumors. J Natl Cancer Inst 2009;101 :8-10), notably because it is collected noninvasively, abundant, and requires minimal preparation. Presently, only one urinary cancer biomarker is clinically applied, PCA3, for detecting prostate cancer (Groskopf J, Aubin SM, Deras IL, et al. APTIMA PCA3 molecular urine test: development of a method to aid in the diagnosis of prostate cancer. Clin Chem 2006;52: 1089-95).
  • urinary biomarkers include modified nucleosides (Henneges C, Bullinger D, Fux R, et al. Prediction of breast cancer by profiling of urinary RNA metabolites using Support Vector Machine-based feature selection. BMC Cancer 2009;9: 104; Hsu WY, Chen WT, Lin WD, et al. Analysis of urinary nucleosides as potential tumor markers in human colorectal cancer by high performance liquid chromatography/electrospray ionization tandem mass spectrometry. Clin Chim Acta 2009;402:31-7; Jeng LB, Lo WY, Hsu WY, et al.
  • Metabolomics is an increasingly popular approach for uncovering new biomarkers for diagnosis (Kim YS, Maruvada P, Milner JA. Metabolomics in biomarker discovery: future uses for cancer prevention. Future Oncol 2008;4:93-102; kind T, Tolstikov V, Fiehn O, Weiss RH. A comprehensive urinary metabolomic approach for identifying kidney cancerr. Anal Biochem 2007;363: 185-95; Matsumura K, Opiekun M, Oka H, et al. Urinary volatile compounds as biomarkers for lung cancer: a proof of principle study using odor signatures in mouse models of lung cancer.
  • Metabolomic studies are unique and powerful because they measure both exogenous (e.g. cigarette smoke constituents) and endogenous molecules from cellular processes reacting to different types of exposures.
  • mass spectrometry is very sensitive and requires only small quantities of biospecimens (Griffin JL. The Cinderella story of metabolic profiling: does metabolomics get to go to the functional genomics ball? Philos Trans R Soc Lond B Biol Sci 2006;361 : 147-61).
  • a recent study has provided proof of principle evidence for the use of metabolomics in smokers that demonstrates the reliability and reproducibility of the assay, and the ability to distinguish levels and smoking status (Hsu PC, Zhou B, Zhao Y, et al. Feasibility of identifying the tobacco-related global metabolome in blood by UPLC-QTOF-MS. J Proteome Res 2012).
  • FIGURE 1 Lung cancer staging staging system.
  • A Definitions used to stage tumors.
  • B Anatomic stage and prognostic groups.
  • FIGURE 2 Quality control assessment in the training set.
  • FIGURE 3 Abundances of three nicotine metabolites (cotinine, nicotine-N' -oxide, and trans-3'-hydroxycotinine), stratified by smoking status, indicating correlation between the self-reported smoking status and nicotine metabolites presence and abundance in the urine.
  • FIGURE 4 Differences in abundance and validation of signals that were top contributors in the classification of patients as lung cancer or healthy controls groups.
  • Untargeted and MSTUS normalized UPLC-MS abundances (mean and standard of the mean (SEM)) are depicted for A) the training set containing 469 cases and 536 controls, B) the validation set comprising 80 cases and 78 controls.
  • Quantitated UPLC-MS/MS abundances (mean and SEM) in C) a subset of the training set containing 92 cases and 106 controls, D) a matched tissue set containing 48 stage I tumors and 48 adjacent non-tumor samples.
  • FIGURE 5 MS-MS validation in comparison to commercially available and synthesized standards of A) Creatine riboside, B) Cortisol sulfate, and C) N-acetyl neuraminic acid. D) MS-MS of un-indentified metabolite 561.3432+. E) Fragmentation pattern depiction of the novel and in-house synthesized compound creatine riboside.
  • FIGURE 6 NMR confirmation of creatine riboside structure. 1 H- 13 C HMBC spectrum of the reaction mixture between ribose and creatine in dmso-d 6 .
  • FIGURE 7 A) Creatine levels (quantitated abundances) are elevated in the tumors of the tissue sample set (48 matched tumor/adjacent non-tumor samples) B) Correlation analysis between creatine riboside and creatine quantitated in tumor tissue samples.
  • FIGURE 8 Association of top four metabolites with lung cancer diagnosis. Signal abundances are dichotomized using the third quartile of healthy control abundances as a cutoff. A) Logistic regression results with reported false discovery rate (FDR) values based on Benjamini-Hochberg method. B) ROC analysis of individual metabolites and their combination in all cases, and in stage I-II cases.
  • FIGURE 10 Diurnal effects on urine metabolites in the training set. Distribution plots depicting relative signal abundances across urine collection times (a- am, p-pm) in A) lung cancer cases and B) population controls.
  • FIGURE 11 Kaplan-Meier survival estimates are depicted for top four biomarkers. Metabolites significantly associated with prognosis in A) all lung cancer patients and B) stage I-II lung cancer patients are shown. The P values reported in the Kaplan-Meier plots reflect the maximum likelihood estimates generated using a univariate Cox model, taking into account left truncation (the lag time between diagnosis and time of urine collection). C) Combination of putative diagnostic and prognostic biomarkers is shown for all cases, and stage I-II cases. Only metabolites that showed statistically significant associations with survival, independent of clinical factors, were combined.
  • FIGURE 14 Overlap between signals that are predictive of lung cancer status (using random forests) in the training set, in samples stratified by race and gender A), and smoking status B).
  • FIGURE 15 Principal Component Analysis (PC A) of colon tumor and non-tumor tissue samples and quality controls (blank, metmix and cocktail containing internal standards).
  • FIGURE 16 Box plots of the relative levels of creatine riboside and NANA in 40 colon cancer matched tumor/non-tumor tissue pairs.
  • FIGURE 17 Logistic regression analysis in liver cancer (Top) and prostate cancer (Bottom).
  • FIGURE 18 Box plots depicting levels of metabolites in (A) liver cancer and (B) prostate cancer compared to their respective population controls. P-values depicted are results of Wilcoxon analysis.
  • Lung cancer remains the leading cause of cancer-related death in both men and women worldwide.
  • the current clinically accepted method for early detection of lung cancer i.e., spiral CT scanning
  • is expensive exposes the patient to high levels of radiation and its use is limited to smokers within a certain age range.
  • current testing procedures result in a high rate of false positives.
  • the present invention provides such methods.
  • the present invention relates to a novel method for detecting cancer by detecting the level of specific biomarkers in an individual. Such a method is inexpensive, requires only a small sample from the patient and can be performed quickly and safely.
  • a method of the present invention may generally be accomplished by determining the levels of one or more specific biomarkers, disclosed herein, within an individual, and determining if such levels are elevated compared to the levels of corresponding biomarkers in a sample from an individual known to be free of cancer. Determining the level of biomarkers of the present invention may also be referred to as determining the signature of the individual. As used herein, a signature refers to the levels of one or more cancer-related biomarkers of the present invention. The inventors have discovered that alterations of these levels relative to the levels of the same one or more biomarkers in individuals known to be free of cancer are indicative of the presence of cancer.
  • a entity or “an” entity refers to one or more of that entity.
  • a nucleic acid molecule refers to one or more nucleic acid molecules.
  • the terms “a”, “an”, “one or more” and “at least one” can be used interchangeably.
  • the terms “comprising”, “including” and “having” can be used interchangeably.
  • the terms individual, subject, patient, and the like are meant to encompass any mammal capable of developing cancer, with a preferred mammal being a human. The terms individual, subject, and patient by themselves do not denote a particular age, sex, race, and the like.
  • the methods of the present invention can be applied to any race of human, including, for example, Caucasian (white), African-American (black), Native American, Native Hawaiian, Hispanic, Latino, Asian, and European.
  • such characteristics may be significant.
  • the significant characteristic(s) e.g., age, sex, race, etc.
  • the term individual encompasses both human and non-human animals.
  • Suitable non-human animals to test for cancer include, but are not limited to companion animals (i.e. pets), food animals, work animals, or zoo animals.
  • Preferred animals include, but are not limited to, cats, dogs, horses, ferrets and other Mustelids, cattle, sheep, swine, and rodents. More preferred animals include cats, dogs, horses and other companion animals, with cats, dogs and horses being even more preferred.
  • the term "companion animal” refers to any animal which a human regards as a pet.
  • a cat refers to any member of the cat family (i.e., Felidae), including domestic cats, wild cats and zoo cats. Examples of cats include, but are not limited to, domestic cats, lions, tigers, leopards, panthers, cougars, bobcats, lynx, jaguars, cheetahs, and servals.
  • a preferred cat is a domestic cat.
  • a dog refers to any member of the family Canidae, including, but not limited to, domestic dogs, wild dogs, foxes, wolves, jackals, and coyotes and other members of the family Canidae. A preferred dog is a domestic dog.
  • a horse refers to any member of the family Equidae.
  • An equid is a hoofed mammal and includes, but is not limited to, domestic horses and wild horses, such as, horses, asses, donkeys, and zebras.
  • Preferred horses include domestic horses, including race horses.
  • the individual being tested may or may not be suspected of having cancer. It will be appreciated by those skilled in the art that some individuals are at higher risk than other individuals for developing cancer. For example, it is known that certain activities and environments increase the risk of developing cancer. Examples of such activities and environments included, but are not limited to smoking, exposure to second-hand smoke, exposure to asbestos, sun-tanning, exposure to radon gas, excessive alcohol consumption, exposure to high levels of radiation, and exposure to chemicals known to be carcinogenic. Thus, in one embodiment, the individual is known to engage in one or more activities that increases the risk for developing cancer. In one embodiment the individual has an increased risk of developing cancer.
  • genes encoding proteins that act as tumor suppressor proteins that act as tumor suppressor proteins. Mutations in these proteins render these proteins inefficient or ineffective resulting in the development of cancer. It is understood by those skilled in the art that a variant gene sequence may be referred to as an allele and that certain alleles are known to be associated with developing cancer. Examples of genes known to be associated with the development of cancer include, but are not limited to P53, APC, RBI, BRCAI, BRCA2, EGFR, KRAS, ALK, RET, KIT, and MET. In one embodiment, the individual carries a mutation or allele that is known to be associated with the development of cancer.
  • cancer refers to diseases in which abnormal cells divide without the appropriate control of cell division and senescence.
  • the cells are able to invade tissues other than those from which the original cancer cells arose.
  • cancer cells may spread to other parts of the body through the blood and lymph systems.
  • cancers are usually named for the organ or type of cell in which they start. For example, a cancer that originates in the colon is called colon cancer; cancer that originates in melanocytes of the skin is called melanoma, etc.
  • cancer may refer to carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., including solid and lymphoid cancers, gastric, kidney cancer, breast cancer, lung cancer(including non-small cell and small cell lung cancer), bladder cancer, colon cancer, ovarian cancer, prostate cancer, pancreas cancer, stomach cancer, brain cancer, head and neck cancers, skin cancer, uterine cancer, testicular cancer, esophageal cancer, liver cancer (including hepatocarcinoma), lymphoma, including non-Hodgkin's lymphomas (e.g., Burkitt's, Small Cell, and Large Cell lymphomas) and Hodgkin's lymphoma, leukemia, and multiple myeloma.
  • the cancer is lung cancer.
  • the cancer is adenocarcinoma.
  • Staging of a cancer is a classification system based on such things as the involvement of lymph nodes and metastasis of the tumor to secondary sites. For example, a new tumor starts out in Stage 0 but as it grows and spreads it progresses through Stages 1-IV.
  • Figure 1A and IB One Example of a staging system is shown in Figure 1A and IB. With regard to the present invention, staging of the cancers disclosed herein was performed according to the method of Edge S., et al, AJCC Cancer Staging Manual.
  • early stages of cancer refer to a cancer that is in Stage 0, Stage I or Stage II.
  • the individual being tested is suspected of being in the early stages of cancer.
  • a biomarker may be described as a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention (Biomarkers Definitions Working Group. Biomarkers and surrogate endpoints: preferred definitions and conceptual framework. Clin Pharmacol Ther. 2001 Mar;69(3):89-95.)
  • biomarker, cancer- marker, cancer-associated biomarker, cancer-associated antigen, and the like refer to a molecule, the level of which is altered in an individual having cancer relative to the level of the same biomarker in an individual known to be free of cancer.
  • a cancer biomarker may be any molecule for which the level of the molecule in an individual having cancer is altered in comparison to the level observed in an individual free of cancer.
  • examples of such molecules include nucleic acid molecules (i.e., RNA and DNA), proteins, lipids, carbohydrates, amino acids, nucleotides and combinations thereof.
  • Preferred biomarkers are those for which the levels of the biomarker may be determined in a quick and efficient manner.
  • the inventors have discovered that the levels of one or more such biomarkers are elevated (i.e., increased) in individuals having cancer.
  • the term elevated refers to an increased level of a biomarker in an individual having cancer compared to the level of biomarker observed in an individual known to be free of cancer (the normal level).
  • the normal level of a biomarker is the level observed in a) a population of individuals known to be free of cancer; and/or 2) the level of biomarker observed in the individual being tested, wherein the level was determined at a time when the individual was known to be free of cancer.
  • a normal level may also be referred to as a base level, control level or reference level.
  • the level of at least one biomarker in an individual having cancer is at least 1.2-fold, at least 1.4-fold, at least 1.6-fold, at least 1.8-fold, at least 2-fold, at least 3 -fold, at least 4-fold, at least 5 -fold, at least 10-fold, at least 15 -fold, at least 20-fold, at least 25 -fold, at least 50-fold or at least 100-fold greater than the normal level of the at least one biomarker.
  • the level of at least one biomarker in an individual having cancer is elevated by at least 20%, at least 30%>, at least 40%>, at least 50%>, at least 100%, at least 200%, at least 300%, at least 400%, at least 500% at least 1000%, at least 2000%, at least 5000% or at least 10,000% over the normal level of the at least one biomarker.
  • one embodiment of the present invention is a method for identifying an individual having cancer, comprising determining the level of at least two biomarkers of the present invention, wherein elevated levels of the at least two biomarkers indicates the presence of cancer.
  • useful biomarkers for identifying individuals having cancer include, but are not limited to, creatine riboside, metabolite 561+, Cortisol sulfate and N-acetylneuraminic acid (NANA).
  • N-acetylneuraminic acid and Cortisol sulfate are shown below and are also described in the database and ontology of Chemical Entities of Biological Interest (ChEBI) of the European Bioinformatics Institute (EBI), which is part of the European Molecular Biology Laboratory.
  • Figure 5E shows the product ion mass spectra obtained by monitoring characteristic fragmentation patterns in multiple reaction monitoring (MRM) mode.
  • MRM multiple reaction monitoring
  • Metabolite 561+ is a glucoronidated lipid, the MS-MS fragmentation patterns of which are shown in Figure 5D. The chemical and physical characteristics of compound 561+ were determined as described in Example 1.
  • One embodiment of the present invention is a method for detecting the presence of cancer in an individual, comprising determining the level of at least two biomarkers selected from the group consisting of creatine riboside, metabolite 561+, Cortisol sulfate and N- acetylneuraminic acid, wherein elevated levels of the at least two biomarkers indicates the presence of cancer.
  • assays using additional biomarkers may lead to improved rates of detection and diagnosis of cancer.
  • one embodiment is a method for detecting the presence of cancer in an individual, comprising determining the level of at least three biomarkers selected from the group consisting of creatine riboside, metabolite 561+, Cortisol sulfate and N-acetylneuraminic acid, wherein elevated levels of the at least three biomarkers indicates the presence of cancer.
  • One embodiment is a method for detecting the presence of cancer in an individual, comprising determining the level of creatine riboside, metabolite 561+, Cortisol sulfate and N-acetylneuraminic acid, wherein elevated levels creatine riboside, metabolite 561+, Cortisol sulfate and N-acetylneuraminic acid indicates the presence of cancer.
  • the cancer is colon cancer.
  • the cancer is lung cancer.
  • the cancer is adenocarcinoma.
  • the cancer is squamous cell carcinoma.
  • the level of one or more biomarkers is determined from a sample obtained, or collected, from an individual to be tested for the presence of cancer.
  • a sample is any specimen obtained from the individual that can be used to measure the level one or more biomarkers.
  • a sample is any specimen obtained from the individual that can be used to measure the level one or more biomarkers selected from the group consisting of creatine riboside, metabolite 561+, Cortisol sulfate and N-acetylneuraminic acid. Examples of useful samples include body fluids and tissue from an individual being tested. A preferred sample is a bodily fluid. Those skilled in the art can readily identify appropriate types of samples.
  • Urine, blood plasma and blood serum are particularly suitable as the sample.
  • Blood plasma plasma
  • blood serum serum
  • Urine samples may also be collected from animals by methods known in the art, including, for example, collecting while the individual is voiding, collecting by catheterization, or by cystocentesis.
  • Urine, plasma, or serum samples may be refrigerated or frozen before assay, but are preferably assayed soon after collection.
  • the level of one or more biomarkers is determined from a plasma or serum sample.
  • the level of one or more biomarkers is determined from a urine sample.
  • the sample may be pre-treated as desired prior to determining the level of biomarkers present in the sample.
  • the sample can be filtered, treated chemically or enzymatically or, if the sample is urine, it may be normalized to a desired specific gravity. Normalizing the sample by appropriate dilution methods known in the art permits quantification of biomarkers independent of the concentration (e.g. specific gravity) of the sample. Any desired specific gravity can be readily selected by those skilled in the art.
  • the level of a biomarker present in a sample may be normalized to another compound present in the sample, such as, for example, hemoglobin level, packed red cell volume or creatinine. Appropriate methods for normalizing a sample are known to those skilled in the art.
  • biomarker levels can be determined by detecting the entire biomarker molecule or by detecting fragments, degradation products or reaction products that are characteristic of the biomarker molecule.
  • determining, measuring or taking a measurement refer to a quantitative or qualitative determination of a property of an entity, for example, quantifying the amount or concentration of a molecule or the activity level of a molecule.
  • concentration or level can refer to an absolute or relative quantity.
  • the level of a biomarker may be reported as a concentration (e,g., ug/ml), it may be reported relative to another value such as a normal value (e.g., 3-fold higher than normal), or it may be reported as a ratio relative to a second molecule (e.g., a biomarker/creatinine ratio of 1.6) .
  • Measuring a molecule may also include determining the absence or presence of the molecule in a sample.
  • any known method of detecting or measuring the level of a biomarker can be used to practice the present invention, so long as the method detects the presence, absence, or level or concentration of the biomarker.
  • methods include, but are not limited to, binding assays, such as an immunological detection assays and non-binding assays (e.g., enzymatic detection assays or assays that detect physical characteristics such as mass).
  • the sample to be tested for the presence, absence or level of a biomarker is contacted with a binding molecule such as, for example, an antibody.
  • a binding molecule such as, for example, an antibody.
  • the term contact, contacted, contacting, and the like refers to the introduction of a sample putatively containing a biomarker to a compound that binds to the biomarker.
  • a biomarker-binding compound is an antibody that selectively binds to the biomarker.
  • other molecules that bind to the biomarker may also be used.
  • the biomarker is a ligand
  • a receptor to which ligand binds can be used as a biomarker-binding compound in assays of the present invention.
  • Appropriate binding molecules for the biomarkers disclosed herein may be determined by those skilled in the art.
  • a biomarker-binding compound complex is formed in a binding assay, such as an immunological assay.
  • complex formation refers to the ability of a biomarker-binding compound to selectively bind to the biomarker in order to form a stable complex that can be detected.
  • selectively, selective, specific, and the like indicate the biomarker-binding compound has a greater affinity for the biomarker than it does for molecules that are unrelated to the biomarker.
  • the terms selectively, selective, specific, and the like indicate that the affinity of the biomarker-binding compound for a biomarker is statistically significantly higher than its affinity for a negative control (e.g., an unrelated molecule, such as, for example, albumin) as measured using a standard assay (e.g., ELISA).
  • a negative control e.g., an unrelated molecule, such as, for example, albumin
  • Detection of the complex can be qualitative, quantitative, or semi-quantitative.
  • Conditions for allowing selective binding and complex formation e.g., appropriate concentrations, buffers, temperatures, reaction times
  • methods to optimize such conditions are known to those skilled in the art.
  • Binding can be measured using a variety of methods standard in the art including, but not limited to, enzyme immunoassays (e.g., ELISA), immunoprecipitations, immunoblot assays and other immunoassays as described, for example, in Sambrook et al., supra, and Harlow, et al, supra. These references also provide examples of complex formation conditions.
  • enzyme immunoassays e.g., ELISA
  • immunoprecipitations e.g., immunoprecipitations
  • immunoblot assays e.g., immunoblot assays and other immunoassays as described, for example, in Sambrook et al., supra, and Harlow, et al, supra. These references also provide examples of complex formation conditions.
  • the biomarker/binding-compound complex also referred to herein as the B/BC complex or simply as the complex
  • the complex can be formed in solution.
  • the complex can be formed while the biomarker-binding compound is immobilized on (e.g., coated onto) a substrate. Immobilization techniques are known to those skilled in the art.
  • Suitable substrate materials include, but are not limited to, plastic, glass, gel, celluloid, fabric, paper, and particulate materials. Examples of substrate materials include, but are not limited to, latex, polystyrene, nylon, nitrocellulose, agarose, cotton, PVDF (polyvinylidene-fluoride), and magnetic resin.
  • Suitable shapes for substrate material include, but are not limited to, a well (e.g., microtiter dish well), a microtiter plate, a dipstick, a strip, a bead, a lateral flow apparatus, a membrane, a filter, a tube, a dish, a celluloid-type matrix, a magnetic particle, and other particulates.
  • Particularly preferred substrates include, for example, an ELISA plate, a dipstick, an immunodot strip, a radioimmunoassay plate, an agarose bead, a plastic bead, a latex bead, a sponge, a cotton thread, a plastic chip, an immunoblot membrane, an immunoblot paper and a flow-through membrane.
  • a substrate such as a particulate
  • a detectable marker for descriptions of examples of substrate materials, see, for example, Kemeny, D. M. (1991) A Practical Guide to ELISA, Pergamon Press, Elmsford, N.Y. pp 33-44, and Price, C. and Newman, D. eds. Principles and Practice of Immunoassay, 2nd edition (1997) Stockton Press, NY, N.Y., both of which are incorporated herein by reference in their entirety.
  • a biomarker-binding compound is immobilized on a substrate, such as the well of a microtiter dish, a dipstick, an immunodot strip, or a lateral flow apparatus.
  • a sample collected from an individual is applied to the substrate and incubated under conditions suitable (i.e., sufficient) to allow the formation of a complex between the binding compound and any biomarker present in the sample.
  • the complex is then detected.
  • detecting the complex refers to identifying the presence of biomarker-binding compound complexed to a biomarker of the present invention. If complexes are formed, the amount of complexes formed can, but need not be, quantified. Complex formation, or selective binding between a biomarker and a biomarker-binding compound, can be measured (i.e., detected, determined) using a variety of methods standard in the art (see, for example, Sambrook et al. supra.), examples of which are disclosed herein.
  • a complex can be detected in a variety of ways including, but not limited to use of one or more of the following assays: an enzyme-linked immunoassay, a competitive enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, a flow-through assay, an agglutination assay, a particulate-based assay (e.g., using particulates such as, but not limited to, magnetic particles or plastic polymers, such as latex or polystyrene beads), an immunoprecipitation assay, a BIACORETM assay (e.g., using colloidal gold), an immunodot assay (e.g., CMG's Immunodot System, Fribourg, Switzerland), and an immunoblot assay (e.g., a western blot), an phosphorescence assay, a flow-through assay, a chromatography as
  • Such assays are well known to those skilled in the art. Some assays, such as agglutination, particulate separation, and immunoprecipitation, can be observed visually (e.g., either by eye or by a machines, such as a densitometer or spectrophotometer) without the need for a detectable marker.
  • conjugation i.e., attachment
  • a detectable marker may be conjugated to the biomarker-binding compound, or reagent, at a site that does not interfere with ability of the compound to bind the biomarker.
  • detectable markers include, but are not limited to, a radioactive label, a fluorescent label, a chemiluminescent label, a chromophoric label, an enzyme label, a phosphorescent label, an electronic label; a metal sol label, a colored bead, a physical label, or a ligand.
  • a ligand refers to a molecule that binds selectively to another molecule.
  • Preferred detectable markers include, but are not limited to, fluorescein, a radioisotope, a phosphatase (e.g., alkaline phosphatase), biotin, avidin, a peroxidase (e.g., horseradish peroxidase), beta- galactosidase, and biotin-related compounds or avidin-related compounds (e.g., streptavidin or IMMUNOPURETM NeutrAvidin).
  • radiolabels may be detected using photographic film or scintillation counters; fluorescent markers may be detected using a photodetector to detect emitted light.
  • Enzymatic markers are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric markers are detected by simply visualizing the colored label.
  • a biomarker/binding compound complex can be detected by contacting a sample with an antibody specific for the binding compound, wherein the antibody is conjugated to a detectable marker.
  • a detectable marker can also be conjugated to an anti-biomarker antibody, or other binding compound.
  • Preferred detectable markers include, but are not limited to, fluorescein, a radioisotope, a phosphatase (e.g., alkaline phosphatase), biotin, avidin, a peroxidase (e.g., horseradish peroxidase), beta-galactosidase, and biotin-related compounds or avidin-related compounds (e.g., streptavidin or IMMUNOPURETM NeutrAvidin).
  • a complex is detected by contacting the complex with an indicator molecule.
  • Suitable indicator molecules include molecules that can bind to the B/BC complex or to the biomarker itself.
  • an indicator molecule can comprise, for example, a biomarker-binding reagent, such as an antibody.
  • indicator molecules that are antibodies include, for example, antibodies reactive with the antibodies from species of animal in which the anti-biomarker antibodies are produced.
  • An indicator molecule itself can be attached to a detectable marker of the present invention.
  • an antibody can be conjugated to biotin, horseradish peroxidase, alkaline phosphatase or fluorescein.
  • the present invention can further comprise one or more layers and/or types of secondary molecules or other binding molecules capable of detecting the presence of an indicator molecule.
  • an untagged (i.e., not conjugated to a detectable marker) secondary antibody that selectively binds to an indicator molecule can be bound to a tagged (i.e., conjugated to a detectable marker) tertiary antibody that selectively binds to the secondary antibody.
  • Suitable secondary antibodies, tertiary antibodies and other secondary or tertiary molecules can be readily selected by those skilled in the art.
  • Preferred tertiary molecules can also be selected by those skilled in the art based upon the characteristics of the secondary molecule. The same strategy can be applied for subsequent layers.
  • the indicator molecule is conjugated to a detectable marker.
  • a developing agent is added, if required, and the substrate is submitted to a detection device for analysis.
  • washing steps are added after one or both complex formation steps in order to remove excess reagents. If such steps are used, they involve conditions known to those skilled in the art such that excess reagents are removed but the complex is retained.
  • One embodiment of the present invention involves the use of a lateral flow assay, examples of which are described in U.S. Patent No. 5,424,193, issued June 13, 1995, to Pronovost et al; U.S. Pat. No. 5,415,994, issued May 16, 1995, by Imrich et al; WO 94/29696, published Dec. 22, 1994, by Miller et al; and WO 94/01775, published Jan. 20, 1994, by Pawlak et al.; all of which are incorporated by reference herein.
  • a lateral flow assay is an example of a single-step assay.
  • a sample is placed in a lateral flow apparatus that includes the following components: (a) a support structure defining a flow path; (b) a labeling reagent comprising a bead conjugated to a specific antibody, the labeling reagent being impregnated within the support structure in a labeling zone; and (c) a capture reagent.
  • the capture reagent is located downstream of the labeling reagent within a capture zone fluidly connected to the labeling zone in such a manner that the labeling reagent can flow from the labeling zone into the capture zone.
  • the support structure comprises a material that does not impede the flow of the beads from the labeling zone to the capture zone. Suitable materials for use as a support structure include ionic (i.e., anionic or cationic) material. Examples of such a material include, but are not limited to, nitrocellulose, PVDF, or carboxymethylcellulose.
  • the support structure defines a flow path that is lateral and is divided into zones, namely a labeling zone and a capture zone.
  • the apparatus can further include a sample receiving zone located along the flow path, preferably upstream of the labeling reagent.
  • the flow path in the support structure is created by contacting a portion of the support structure downstream of the capture zone, preferably at the end of the flow path, to an absorbent capable of absorbing excess liquid from the labeling and capture zones.
  • a lateral flow apparatus used to detect a biomarker includes: (a) a support structure defining a flow path; (b) a labeling reagent comprising an anti- biomarker antibody as described above, the labeling reagent impregnated within the support structure in a labeling zone; and (c) a capture reagent, the capture reagent being located downstream of the labeling reagent within a capture zone fluidly connected to the labeling zone in such a manner that the labeling reagent can flow from the labeling zone into the capture zone.
  • the apparatus preferably also includes a sample receiving zone located along the flow path, preferably upstream of the labeling reagent.
  • the apparatus preferably also includes an absorbent located at the end of the flow path.
  • One preferred embodiment includes a capture reagent comprising a biomarker binding compound.
  • Dipsticks are constructed in a variety of ways that partly depend on the way in which they will be used. They may be held directly in a sample (e.g., a urine stream), dipped directly in sample contained in a collection vessel, or have sample applied to a strip contained in a plastic cassette or platform.
  • a sample e.g., a urine stream
  • dipped directly in sample contained in a collection vessel or have sample applied to a strip contained in a plastic cassette or platform.
  • dipstick is a "flow-through” device, an example of which is a heterogenous immunometric assay system based on a capture antibody immobilized onto a membrane attached to an absorbent reservoir
  • a "bead” refers to a particulate substrate composed of a matrix such as latex or polystyrene, which can be covalently or non-covalently cross-linked to a detection molecule.
  • a preferred embodiment of the "dipstick” assay is an immunometric system, described in U.S. Pat. No. 5,656,502, issued on Aug. 12, 1997, to MacKay and Fredrickson, and U.S. Pat. No. 6,001,658, issued Dec. 14, 1999 to Fredrickson, both incorporated herein by reference. Particularly preferred is an IMMUNODIPTM device available from Diagnostic Chemicals Ltd., PEI, CA.
  • various methods of detecting and/or determining the level of a biomarker include, but are not limited to, refractive index spectroscopy (RI), ultra-violet spectroscopy (UV), fluorescence analysis, electrochemical analysis, radiochemical analysis, near-infrared spectroscopy (near-IR), infrared (IR) spectroscopy, nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS), mass spectrometry, pyrolysis mass spectrometry, nephelometry, dispersive Raman spectroscopy, gas chromatography, liquid chromatography, gas chromatography combined with mass spectrometry, liquid chromatography combined with mass spectrometry, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) combined with mass spectrometry, ion spray spectroscopy combined with mass spectrometry, capillary electrophoresis, colorimetry and surface plasmon resonance (such
  • biomarkers can be measured using the above-mentioned detection methods, or other methods known to the skilled artisan.
  • Other biomarkers can be similarly detected using reagents that are specifically designed or tailored to detect them.
  • the level may be compared to the normal level of the same biomarker and a determination of whether or not the individual has cancer can be made.
  • elevated levels of one or more of the biomarkers disclosed herein indicate the individual has cancer.
  • an elevated level of at least two compounds selected from the group consisting of creatine riboside, metabolite 561+, Cortisol sulfate and N-acetylneuraminic acid in a sample obtained from the individual indicates the presence of cancer.
  • Comparison of the level of biomarker in the individual being tested to the normal level may be performed by any method that allows the detection of the level of a biomarker.
  • a corresponding immunoassay may be performed using a sample from a normal individual and the numeric results from the assays compared.
  • Assay of the normal sample may be performed at the same time as the test sample or it may be performed prior to, or after, the level of biomarker in the test sample is determined.
  • Biomarkers levels from the individual being tested can also be compared to a historical normal value, which is a value obtained from one or more cancer- free individuals over time. Comparison of the results may be performed visually or they may be performed by a machine (e.g., computer).
  • the output of such comparison may be a numeric value, such as the difference between values, or it may be a qualitative result, such as a yes or no with regard to the presence of cancer. Similar methods of comparison may be performed using any of the detection methods disclosed herein (e.g., mass spectrometry).
  • the number of biomarkers chosen for measurement, and the sample used to determine the level of the biomarker will vary. For example, in some instances, determination of the level of a single biomarker is sufficient for the detection of cancer.
  • useful, single biomarkers include, but are not limited to, creatine riboside and N-acetylneuraminic acid (NANA).
  • NANA N-acetylneuraminic acid
  • one embodiment of the present invention is a method to detect the presence of cancer in an individual, the method comprising determining the level of one or more biomarkers selected from the group consisting of creatine riboside and NANA in a sample from the individual, wherein elevated levels of creatine riboside or NANA indicates the presence of cancer in the individual.
  • the biomarker being measured is creatine riboside. In one embodiment, the biomarker being measured is NANA. In one embodiment, the level of biomarker is determined from a tissue sample. In one embodiment, the level of biomarker is determined from at least one body fluid selected from the group consisting of plasma, serum and urine. In one embodiment, the body fluid is urine. In one embodiment the cancer is colon cancer. In one embodiment, the cancer is lung cancer. In one embodiment, the cancer is adenocarcinoma.
  • one embodiment of the present invention is a method to detect the presence of lung cancer in an individual, the method comprising determining the level of Cortisol sulfate in body fluid obtained from the individual, wherein elevated levels of Cortisol sulfate indicates the presence of cancer in the individual.
  • the body fluid may be plasma, serum or urine.
  • the cancer is adenocarcinoma. In one embodiment, the cancer is squamous cell carcinoma.
  • elevated levels of a specific biomarker in a particular bodily fluid are indicative of the presence of specific types of cancers.
  • the inventors have discovered that elevated levels of N-acetylneuraminic acid in the urine are indicative of lung cancer.
  • one embodiment of the present invention is a method to detect the presence of cancer in an individual, the method comprising determining the level of N- acetylneuraminic acid in urine from the individual, wherein elevated levels of urinary N- acetylneuraminic acid indicates the presence of lung cancer in the individual.
  • the cancer is an adenocarcinoma.
  • the cancer is squamous cell carcinoma.
  • one embodiment of the present invention is a method for identifying an individual having cancer, the method comprising determining the level of at least two biomarkers in a body fluid obtained from an individual, wherein the at least two biomarkers are selected from the group consisting of creatine riboside, metabolite 561+, Cortisol sulfate and N-acetylneuraminic acid, and wherein elevated levels of the at least two biomarkers identifies the individual as having cancer.
  • One embodiment of the present invention is a method for identifying an individual having cancer, the method comprising determining the level of at least three biomarkers in a body fluid obtained from an individual, wherein the at least three biomarkers are selected from the group consisting of creatine riboside, metabolite 561+, Cortisol sulfate and N- acetylneuraminic acid, and wherein elevated levels of the at least three biomarkers identifies the individual as having cancer.
  • One embodiment is a method for identifying an individual having cancer, comprising determining the level of creatine riboside, metabolite 561+, Cortisol sulfate and N-acetylneuraminic acid, wherein elevated levels creatine riboside, metabolite 561+, Cortisol sulfate and N-acetylneuraminic acid identify the individual as having cancer.
  • One embodiment of the present invention is a method to identify an individual having cancer, the method comprising determining the level of one or more biomarkers selected from the group consisting of creatine riboside and NANA, wherein elevated levels of creatine riboside and/or NANA identifies the individual as having cancer.
  • the cancer is colon cancer.
  • the cancer is lung cancer.
  • the cancer is adenocarcinoma.
  • the cancer is squamous cell carcinoma.
  • One embodiment of the present invention is a method to identify an individual having lung cancer, the method comprising determining the level of Cortisol sulfate in a body fluid obtained from an individual, wherein elevated levels of Cortisol sulfate identify the individual as having lung cancer.
  • One embodiment of the present invention is a method to identify an individual having lung cancer, the method comprising determining the level of N- acetylneuraminic acid in urine obtained from an individual, wherein elevated levels of urinary N-acetylneuraminic acid identifies the individual as having lung cancer.
  • a treatment or therapy for cancer refers to any treatment or therapy that is intended to reduce the cancer load or prevent the cancer load from increasing. Examples of treatments include, but are not limited to, surgery, chemotherapy, biotherapy and radiation.
  • the term cancer load may refer to the mass, size or number of cancer cells present in a patient.
  • the at least two biomarkers are selected from the group consisting of creatine riboside, metabolite 561+, Cortisol sulfate and N-acetylneuraminic acid.
  • the level of at least three biomarkers are determined, wherein the biomarkers are selected from the group consisting of creatine riboside, metabolite 561+, Cortisol sulfate and N- acetylneuraminic acid.
  • the level of creatine riboside, metabolite 561+, Cortisol sulfate and N-acetylneuraminic acid are detected.
  • one embodiment of the present invention is a method for monitoring the efficacy of a cancer treatment, the method comprising:
  • efficacy of treatment for cancer is monitored by determining the level of one or more biomarkers selected from the group consisting of creatine riboside and N-acetylneuraminic acid (NANA). In one embodiment, efficacy of treatment for cancer is monitored by determining the level of at least two biomarkers selected from the group consisting of creatine riboside, metabolite 561+, Cortisol sulfate and N- acetylneuraminic acid in body fluid obtained from the individual.
  • efficacy of treatment for lung cancer is monitored by determining the level of Cortisol sulfate in a sample of body fluid from the individual. In one embodiment, efficacy of treatment for lung cancer is monitored by determining the level of N-acetylneuraminic acid in urine from the individual.
  • one embodiment of the present invention is a method for predicting the prognosis of an individual having cancer, the method comprising determining the level of at least one biomarker selected from the group consisting of creatine riboside and metabolite 561+, wherein an elevated level of the at least one biomarker is indicative of the prognosis of the individual.
  • the levels of creatine riboside and metabolite 561+ are determined.
  • the at least one biomarker level is determined when the cancer is in an early stage.
  • an elevated level of the at least one biomarker indicates a reduced survival time relative to a cancer patient in whom the level of the at least one biomarker is not elevated.
  • elevated levels of creatine riboside and metabolite 561+ indicate a reduced survival time relative to a cancer patient in whom the levels of creatine riboside and metabolite 561+ are not elevated.
  • biomarkers of the present invention may be improved when the levels of such biomarkers are measured in combination with the level or presence of other, known cancer biomarkers.
  • the known biomarker may be any molecule for which an association between the level or presence of the molecule and a diagnosis of cancer ( or a prognosis related thereto) has been established.
  • known biomarkers to measure include, but are not limited to, proteins, nucleic acid molecules, lipids, carbohydrates and combinations thereof.
  • One type of known biomarker to measure in combination with one or more biomarker of the present invention is a microRNA (miRNA).
  • MicroRNAs are small, non-coding RNAs, that have been shown to regulate epigenetic phenomena (Lee, RC, Feinbaum RL, Ambros, V.
  • the C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complimentarity to lin-14. Cell 993; 75(5):843-54).
  • These small RNA molecules which are usually 18-25 nucleotides in size, repress the translation of target genes by complimentary binding to their 3 'UTR sequence (Carthew, RW, Sontheimer EJ. Origins and Mechanisms of miRNAs and siRNAs. Cell 2009; 136(4):642-55).
  • adenocarcinoma a retrospective analysis of three cohorts. Clin Cancer Res 2011; 17(7): 1875- 82; Park JK, Lee EJ, Esau C, et al. Antisense inhibition of microRNA-21 or -221 arrests cell cycle, induces apoptosis, and sensitizes the effects of gemcitabine in pancreatic
  • MicroRNA-21 post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer.
  • Oncogene 2008;27(15):2128-36) In fact, a number of miRs have been found to be deregulated in several types of cancers (Park JK, Lee EJ, Esau C, et al.
  • Antisense inhibition of microRNA-21 or -221 arrests cell cycle, induces apoptosis, and sensitizes the effects of gemcitabine in pancreatic adenocarcinoma.
  • MicroRNA-21 post- transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion
  • MicroRNA-21 targets a network of key tumor- suppressive pathways in glioblastoma cells. Cancer Res 2008;68(19):8164-72; Meng F, Henson R, Wehbe-Janek H, et al. MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology 2007;133(2):647-58;
  • a microRNA expression signature of human solid tumors defines cancer gene targets.
  • Proc Natl Acad Sci U S A 2006;103(7):2257-61; Takahashi Y, Forrest AR, Maeno E, et al. MiR-107 and MiR-185 can induce cell cycle arrest in human non small cell lung cancer cell lines.
  • mir-21 has been found to be up-regulated in most cancer sites, including lung, pancreas, liver and colon (Saito M, Schetter AJ, Mollerup S, et al.
  • MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology 2007;133(2):647-58). An increase in miR-21 expression has been associated with poor prognosis in lung cancer patients (Saito M, Schetter AJ, Mollerup S, et al. The association of microRNA expression with prognosis and progression in early-stage, non-small cell lung adenocarcinoma: a retrospective analysis of three cohorts. Clin Cancer Res 2011 ; 17(7): 1875-82; Markou A, Tsaroucha EG, Kaklamanis L, et al. Prognostic value of mature microRNA-21 and microRNA-205 overexpression in non-small cell lung cancer by quantitative real-time RT-PCR.
  • the presence of cancer is detected by measuring the level of one or more biomarkers in conjunction with the level of one or more microRNAs.
  • Methods of measuring miRNA levels are known to those skilled in the art.
  • the level of miRNA may or may not be determined at the same time as the level of the one or more biomarker of the present invention.
  • the level of miRNA may or may not be determined using the sample from which the level of the one or more biomarker is determined.
  • the cancer is lung cancer.
  • the cancer is adenocarcinoma or squamous cell carcinoma.
  • the one or more biomarker is selected from the group consisting of creatine riboside, metabolite 561+, Cortisol sulfate and N-acetylneuraminic acid.
  • kits useful for practicing the disclosed methods of the present invention are kits useful for practicing the disclosed methods of the present invention.
  • a kit for identifying an individual having cancer in accordance with the present invention, the kit comprising i) reagents for selectively detecting the presence, absence or level of, at least one or more biomarkers in a sample obtained from the subject and ii) instructions for using the kit.
  • a kit for detecting cancer or identifying an individual having cancer in accordance with the present invention, the kit comprising i) reagents for selectively detecting the presence, absence or level of, at least one biomarker of the present invention in a sample obtained from the subject and ii) instructions for using the kit.
  • Kits of the present invention will contain at least some of the reagents required to determine the presence, absence or level of biomarkers of the present invention.
  • Reagents for kits of the present invention can include, but are not limited to, isolated biomarkers of the present invention, and compounds that bind biomarkers of the present invention (e.g., an antibody that selectively binds to a biomarker of the present invention).
  • the biomarker protein and/or the biomarker-binding compound may be fixed to a solid substrate.
  • the kits may further comprise control proteins.
  • One skilled in the art will, without undue experiments, be able to select the necessary reagents from the disclosure herein, in accordance with the usual requirements.
  • Reagents of the kit may also comprise a molecular label or tag.
  • Kits of the present invention can also comprise various reagents, such as buffers, necessary to practice the methods of the invention, as known in the art. These reagents or buffers may, for example, be useful to extract and/or purify biomarkers from the biological sample obtained from the subject.
  • the kit may also comprise all the necessary material such as microcentrifuge tubes necessary to practice the methods of the invention. Examples
  • Urine samples from 469 lung cancer patients prior to treatment and 536 population controls collected from 1998 to 2007 from the greater Baltimore, Maryland area were used as a training set. Patients were recruited from pathology departments in seven hospitals: Baltimore Veterans Administration Medical Center, Bon Secours Hospital, Harbor Hospital Center, Yale Hospital, Johns Hopkins Bayview Medical Center, The Johns Hopkins Hospital, and University of Maryland Medical Center. Population controls were identified from the Department of Motor Vehicles lists and frequency-matched to cases by age, gender, and ethnicity (Zheng YL, Lloiso CA, Yu Z, et al. Bleomycin-induced chromosome breaks as a risk marker for lung cancer: a case-control study with population and hospital controls.
  • Urine samples were analyzed using a quadrupole time-of-flight (QTOF) mass spectrometer (Premier, Waters), in positive (ESI+) and negative (ESI-) electrospray ionization modes, using a 50 x 2.1 mm Acquity 1.7 ⁇ CI 8 column (Waters Corp, Millford, MA). Briefly, urine samples were diluted with an equal volume of 50% aqueous acetonitrile containing debrisoquine (ESI+ internal standard) and 4-nitrobenzoic acid (ESI- internal standard). Samples were centrifuged at 14,000 x g for 20 minutes at 4°C to precipitate proteins.
  • QTOF quadrupole time-of-flight
  • the eluent was introduced by electrospray ionization into the QTOF mass spectrometer (Premier, Waters) operating in positive (ESI+) or negative (ESI-) ionization mode.
  • the capillary and sampling cone voltages were set to 3,000 and 30 V, respectively.
  • Source and desolvation temperatures were set to 120 °C and 350 °C, respectively, and the cone and desolvation gas flows were set to 50.0 and 650.0 L/h, respectively.
  • sulfadimethoxine at a concentration of 300 pg/ ⁇ in 50%
  • aqueous acetonitrile was used as a lock mass and injected at a rate of 50 ⁇ /min.
  • XCMS processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification.
  • Retention times were aligned with one iteration of the rector() function using the loess method, which performs smoothing of the retention time deviations for every time point in each sample.
  • the validation set data from 158 samples was also processed using XCMS R package following the same parameters outlined for the original set.
  • Metabolite identity was confirmed using commercially available and in-house synthesized standards, by comparison of retention time, product ion mass spectra and by monitoring characteristic fragmentation patterns in multiple reaction monitoring (MRM) mode.
  • MRM multiple reaction monitoring
  • Cortisol-21 -sulfate was purified from the reaction mixture using anion exchange resin as described earlier [ref. Mumma RO, Hoiberg CP, Weber WW, 2nd. Preparation of sulfate esters.
  • the structure of creatine riboside was determined using UPLC-QTOF MS/MS.
  • the gradient elution was performed over 10 min using: 1-60% B in 4 min (0.4 ml/min), 60-80% B at 8 min (0.4 ml/min), holding at 80% B up to 8.5 min (0.3 ml/min), bringing back to 1% B at 8.8 min and holding at 1% until end (0.3 ml/min).
  • the column was re-equilibrated with 99% A at the end of each run prior to injection of next sample.
  • the eluent was introduced via electrospray into a Synapt G2S mass spectrometer (Waters) and tandem MS was generated for 264.1196+ corresponding to the predicted m/z for creatine riboside.
  • HMBC is a 2D NMR experiment which gives the long range (2- or 3 -bonds) couplings between protons and carbons.
  • Figure 6 there is a cross peak between the anomeric sugar proton of ribose at 4.61 ppm and the carbon at 156.19 ppm of creatine, indicating the covalent attachment between the two units.
  • This Example demonstrates the ability of methodology of the present invention to predict the smoking status and/or the cancer status of test individuals.
  • the 3 most highly associated metabolites are cotinine, nicotine -N' -oxide, and trans-3'- hydroxycotinine, known nicotine metabolites. This finding establishes the utility of random forests-based classification approach to find diagnostic metabolites of lung cancer.
  • African- American menthol and nonmenthol smokers differences in smoking and cessation experiences.
  • J Natl Med Assoc 2004;96: 1208-11 including the preference for menthol cigarettes amongst African Americans Health 1991;81 : 1483-6; Okuyemi KS, Ebersole- Robinson M, Vietnameser N, Ahluwalia JS. African- American menthol and nonmenthol smokers: differences in smoking and cessation experiences.
  • Proportion of samples that were accurately categorized as lung cancer cases or controls using optimal variables were as follows: 78.1% using all samples, 77.7% for Caucasian males, 78.6% for Caucasian females, 84.9% for African American males, and 82.3% for African American females. AS shown in Table 3 below, true positive and true negative rates ranged from 77.1- 84.9 and 63.2-81.7, respectively.
  • This Example demonstrates the presence of the identified biomarkers in tumor tissue.
  • Tumor and matched adjacent normal tissues were pulverized by cryogenic grinding (Cryomill®, Retsch GmbH, Haan, Germany) using a 5 mm stainless steel ball. Average sample weight was 15mg. A monophasic mixture of ice-cold chloroform:methanol:water (2:5:2, v:v:v) was used for extraction. Samples were centrifuged at 14,000xg for 15 minutes at 4°C, and reconstituted in 70% aqueous acetonitrile before they were injected onto the Xevo TQMS system.
  • Creatine riboside and N-acetylneuraminic acid were significantly more abundant in tumor tissue compared to adjacent normal tissue, as assessed in 48 stage I adenocarcinoma and squamous cell carcinoma patients ( Figure 4D). Creatine was also elevated in the tumor compared to adjacent normal tissue, and correlates with creatine riboside ( Figure 7).
  • This Example illustrates an assessment of the contribution of each metabolite to lung cancer.
  • This Example illustrates an assessment of the association between levels of biomarkers of the present invention and prognosis.
  • This Example compares the levels of biomarkers of the present invention present in colon tumor tissue with the levels observed in non-cancerous tissue.
  • Average sample weight was lOmg.
  • Each tissue sample was processed twice, the first method using Cryogenic grinding, and the second method using tissue homogenization at room temperature.
  • the first method consisted of cryogenic pulverization of tissue at— 200 °C
  • This Example examines the ability of the levels of biomarkers of the present invention in urine to diagnose and predict the prognosis of lung cancer.
  • HILIC chromatography

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Abstract

La présente invention concerne des méthodes et des matériaux utilisables en vue du diagnostic du cancer chez un sujet, ledit diagnostic étant établi sur la base d'un échantillon de tissu, de sang ou d'urine provenant du patient. De façon plus précise, la méthode selon l'invention implique de déterminer le niveau d'un ou plusieurs métabolites choisis dans le groupe constitué de la créatine riboside, du métabolite 561+, du sulfate de cortisol et de l'acide N-acétylneuraminique. La présente invention concerne également une méthode de détermination du pronostic pour un patient cancéreux, ladite méthode consistant à déterminer le niveau d'un ou plusieurs métabolites choisis dans le groupe constitué de la créatine riboside, du métabolite 561+, du sulfate de cortisol et de l'acide N-acétylneuraminique. L'invention concerne également des nécessaires permettant de détecter un cancer ou de déterminer un pronostic pour un patient cancéreux.
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WO2023108166A2 (fr) * 2021-12-10 2023-06-15 The Johns Hopkins University Biomarqueurs pour détecter un cancer de la prostate agressif à partir de formes indolentes et leur traitement
WO2023196571A1 (fr) * 2022-04-08 2023-10-12 The Johns Hopkins University Détection d'un cancer hypermétabolique par apprentissage automatique sur la base de spectres de résonance magnétique nucléaire

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WO2015006657A8 (fr) 2015-03-12
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US20160169899A1 (en) 2016-06-16
US11555818B2 (en) 2023-01-17

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